Drynaria extractions for treating osteoporosis and their extraction process

ABSTRACT

Rhizoma Drynariae  extract (RDE) is used as therapeutical agent or in the preparation for Osteoporosis, which is characterized in containing over 30 percent of total flavonoids. Furthermore naringin contained in flavonoids is more than 30 percent and less than 100 percent. The RDE is applied for the therapy of osteoporosis or used for producing drugs treating osteoporosis. And methods of extracting the same are also related.

FIELD OF THE INVENTION

[0001] The present invention relates to Rhizoma Drynariae Extract (RDE),more specifically to a RDE for the treatment of osteoporosis, and thepreparation process of such and the use thereof in the treatment ofosteoporosis. The present invention is also related to the method oftreating osteoporosis with the extract.

BACKGROUND OF THE INVENTION

[0002]Rhizoma Drynariae is the rhizome of Drynaria fortunei (Kunze) J.Sm or D. baronii (Chist) Diels, which is used for the treatment of bonefracture for a long time (Zhong Yao Da Ci Dian (Lexicon of TraditionalChinese Medicine), 1658-1660, Shanghai People's Press, ShangHai, 1979).Ma Ke-Chang et al., studied the influence of Rhizoma Drynariae on ratOsteoporosis model, the results showed that Rhizoma Drynariae extractpartically restrains the glucocorticoid-induced bone loss (Ma Ke-Changet al., Zhong Yi Zheng Gu (Bone Setting) 1992, 4, 3), but the activecomponent therein is unknown. Additionally, Zhou Tong-Shui et al.reported that the effective component of Rhizoma Drynariae for treatingbone injury is naringin and analogues thereof (Zhou Tong-Shui, et. al.,Zhong Cao Yao (Chinese Herbal Medicine) 1994, 25, 249). And they alsoperformed assaying in crude plant of Rhizoma Drynariae (Zhou Tong-shuiet al., Zhong Guo Yao Ke Da Xue Xue Bao (Journal of China PharmaceuticalUniversity), 1996, 27, (9), 540), but they didn't show the relationshipbetween the above-mentioned active components and the effect toOsteoporosis thereof.

[0003] Wu Ying-Pi et al. disclosed a method of extracting naringin fromCitrus grandis Osbeck var. tomentosa Hort, aiming to obtain the singlecomponent of naringin (Wu Ying-Pi et al., Zhong Cao Yao, 1988, 19, 452)only. Therefore, it is unknown whether or not such method can be fittedfor other medicinal materials.

SUMMARY OF THE INVENTION

[0004] The object of the present invention is to provide RhizomaDrynariae extract (herein after also referred as RDE), which isavailable for the treatment of Osteoporosis.

[0005] The other object is to provide a composition comprising RDE andthe use of which for the treatment of osteoporosis.

[0006] Another object is to provide a process of preparation of RDE.

[0007] One further object of the present invention is to provide acomposition comprising naringin for the treatment of osteoporosis, andthe treatment method of osteoporosis with the composition.

[0008] The technical embodiments of the present invention are asfollowing:

[0009] 1. An extract from Rhizoma Drynariae, wherein the content oftotal flavonoids, based on the weight of the extract, is 30% or more,and the content of naringin thereof, based on the weight of totalflavonoids, is 30% or more, and less than 100 percent.

[0010] 2. The extract according to 1, wherein the content of totalflavonoids is 50% or more.

[0011] 3. The extract according to 2, wherein the content of naringin is90% or less.

[0012] 4. A pharmaceutical composition comprises the extract of 1.

[0013] 5. The composition according to 4, which is used for thetreatment of osteoporosis.

[0014] 6. Use of the extract according to 1 in the preparation ofmedicaments for the treatment of osteoporosis.

[0015] 7. Use of the extract according to 1 for the treatment ofosteoporosis.

[0016] 8. A preparation process of the extract according to 1,comprising:

[0017] 1) extracting Rhizoma Drynariae with water, alcohol or a mixturethereof;

[0018] 2) adsorbing of the extractant with resin; and

[0019] 3) elution of the resin which has absorbed the extractant withwater, alcohol or a mixture thereof.

[0020] 9. The preparation process according to 8, wherein the extractionis conducted with water.

[0021] 10. The preparation process according to 8, wherein theextraction is conducted with alcohol/water mixture.

[0022] 11. The preparation process according to 8 or 10, wherein thecontent of alcohol in the alcohol/water mixture is 40-90% (w/w).

[0023] 12. The preparation process according to 8, 10 or 11, wherein thealcohol is selected from methanol and ethanol.

[0024] 13. The preparation process according to 12, wherein the alcoholis ethanol.

[0025] 14. The preparation process according to 13, wherein the resin ismacropore adsorption resin.

[0026] 15. The preparation process according to 8 or 14, wherein saidresin is washed by water after adsorption of the extractant, before theelution with alcohol or alcohol/water mixture.

[0027] 16. The preparation process according to 15, wherein theextraction is conducted with alcohol/water mixture.

[0028] 17. The preparation process according to 15 or 16, wherein thecontent of alcohol in the alcohol/water mixture is 40-90% (w/w).

[0029] 18. The preparation process according to 15, 16 or 17, whereinthe alcohol is selected from methanol and ethanol.

[0030] 19. The preparation process according to 18, wherein the alcoholis ethanol.

BRIEF DESCRIPTION OF THE FIGURES

[0031]FIG. 1 shows HPLC chromatograph of standard naringin as control.

[0032]FIG. 2 shows the UV spectrographs of standard naringin, thesolution of the extract of Example 1 and blank solution.

[0033]FIG. 3 shows HPLC chromatograph of the extract of Example 1.

[0034]FIG. 4 shows the comparison of TLC chromatographs of the solutionsof the extracts from different processes.

[0035]FIG. 5 shows HPLC chromatograph of the extract of Example 3.

[0036]FIG. 6 shows HPLC chromatograph of the extract of Example 4.

DETAILED DESCRIPTION OF THE INVENTION

[0037] In the following descriptions of the present invention,percentage represents weight percentage, unless specifically identified.

[0038] In the present invention, Rhizoma Drynariae may be the rhizome orthe whole plant of the plants from the genus Drynaria (Bory) J. Sm,Polypodiaceae S. F. Gary, such as Drynaria fortunei (Kunze) J. Sm, D.baronii (Chist) Diels, D. sinica Diels, and D. delavayi Chirst, et al.,or Davallia mariesii Moore ex Bak from the genus Davallia Sm, and thelike. In the present invention, plants used as Rhizoma Drynariae may befrom one single species or a mixture of multiple species as describedabove, and is not limited.

[0039] The inventors have conducted an intensive investigation and foundthat particular extract from Rhizoma Drynariae or the single compoundnaringin have the activity of improving osteoporosis. Among the extractpossessing activity against osteoporosis, the content of totalflavonoids is always more than 30%, and content of naringin in theflavonoids is more than 30%. The compound naringin per se also has theactivity of improving osteoporosis, whereas the efficiency thereof islower than the active extracts described above.

[0040] In the present invention, the total flavonoids contained in RDEshould be 30% (wt %) or more, preferably 40% or more, more preferably45% or more, and most preferably 50% or more. Though there is noparticular upper limit for the content of total flavonoids, which can beranged from 30% to 100% in the extract, however, while considering thebalance of the choice of process and cost, as long as content of totalflavonoids is 50% or more, the object of this present invention can bewell achieved. If the content is upper than 90%, it does not only costmore, but extra procedure is also needed. Therefore the most preferabletotal flavonoids content ranged from 50% to 90%.

[0041] According to the present method, the RDE of present inventionalso comprises polyphenol compounds. Although there is no evidence forpolyphenol compounds against osteoporosis, but the extract of presentinvention may preferrably contain polyphenols, for polyphenols have theactivity of anti-oxidization, therefore the polyphenol compounds may behelpful for the present extract in the use of preperation ofpharmaceuticals. The content of polyphenols may vary depending ondifferent processes, but the contents are normally ranging from 10% to50%.

[0042] In the present invention, naringin's content should be 30% ormore, preferably 40% or more, and more preferably 50% or more, withrespect to total flavonoids, but naringin shouldn't be the onlyflavonoid of total flavonoids, preferably no more than 90%, and morepreferably no more than 80%.

[0043] We have extracted the flavonoids containing extract of RhizomaDrynariae (as defined above). The results of experiments and clinicaltrials have shown that the Rhizoma Drynariae extract produced by theprocess of present invention has reliable effects on osteoporosis.

[0044] Although it is not proved by any known theories, but it isreasonable that the activity of anti-osteoporosis of the present extractis not only rooted in naringin, but also some other components which mayhave the synergic effects with naringin. Under the limitations ofpresent invention, such components co-present with naringin and show theactivity of anti-osteoporosis.

[0045] The producing process of Rhizoma Drynariae extract is discussedherein after.

[0046] A typical producing process may comprise the following steps:[Crude drug pulverizing]→water/alcohol decoction→resin adsorption→(waterwashing)→alcohol/water elution—post procession

[0047] Among the process, means to pulverize Rhizoma Drynariae arecommon processes to those skilled in the art and are not limited. If thegranularity of powder is too coarse, the active components will not besufficiently extracted; if the granularity is too fine, the separationof extracted solution after decoction may be difficult. Such can bereadily determined by the experience of those skilled in the art, andwill not be limited.

[0048] In order to extract effective component, soaking extraction bywater and/or alcohol may also be adopted. However, since the effectivecomponent may not be lixiviated completely, heat extraction ispreferred, the heating temperature may range from room temperature tothe boiling point of extracting solvent.

[0049] The temperature in heat extraction is preferably conducted at theboiling point of extracting solvent (reflux).

[0050] In case when the crude drug is extracted with water along, theextraction is conducted under 100° C. for 0.5-3 hrs and repeated for 2-4times, while the volume of water is 5-20 folds by weight of crude drugeach time. In case when extraction is conducted with alcohol or alcoholaqueous solution under reflux temperature, the crude drug is extractedfor 0.5-3hrs and repeated for 2-4 times, while the volume of solvent is5-20 folds by weight of crude drug each time. The alcohol may bemethanol or ethanol, and ethanol is preferred from the viewpoint ofsafety. When adopted, the concentration of ethanol is preferably in therange of 20-90%. And all the data mentioned above are for referenceonly. It can be modified based on the experience of person of the art,and it should be understood that such changes are not beyond the scopeof the present invention.

[0051] If desired, the extractant obtained may firstly be filtrated,then adsorbed by resin, which may be macropore adsorption resin, such asD₁₀₁ resin (manufactured by Nankai University Resin Factory, Tianjin),AB-8 resin (manufactured by Tianjin Bone-gel Factory), WLD resin(manufactured by the Chinese Traditional Medicine Institute of SichuanProvince) and CAD-40 resin (manufactured by Huabei Pharmaceuticals),etc. The method of this present invention is not limited by the model ofresins. The detailed description of resin adsorption is described inCN1072089A, and referred herein with the present invention andincorporated as a part of it.

[0052] According to the research of the present inventors, WLD resin hasthe highest adsorption capability among the resins mentioned above, andis much easier to be eluted. So use of WLD resin as adsorbent ispreferred. The ratio of the amount of resin to crude drug may range from0.5-2:1 (w/w), preferably 0.5-1.5:1, most preferably about 1:1, whichcan be well determined by those of the art. If the ratio is less than0.5:1, it is likely to adsorb incompletely. If it is more than 1.5:1,the adsorbed flavonoid may not increase much more, and may causeeconomical disadvantages.

[0053] While employing adsorption resin, according to the commonknowledge well known by those of the art, the resin should be pretreatedvia known methods before using, for example the resin might bepretreated as following:

[0054] packing the resin in a column,

[0055] rinsing the column with ethanol-conc. HCl (1:1) until theeffluent fluid is clear upon dilution with a equivolume of water, then

[0056] flushing the column with hot water (about 80° C.) ten-foldsvolume of the resin column, after that,

[0057] rinsing the column with 2% sodium hydroxide -equivolume of theresin column, and finally

[0058] rinsing with water until the effluent fluid become neutral.

[0059] The resin is then reverse-rinsed with water to loose, and thenupload the solution to be eluted.

[0060] During absorption, the flow-rate should be controlled to ensurecomplete adsorption. Generally, at the time of industrial manufaction,while a column (φ 360 mm) packed with 100 kg of resin is employed, theadsorption may be ensured to be complete if the flow-rate be adjusted to10-20 L·min⁻¹. In such case, if the flow-rate is more than 20 L·min⁻¹,it is hard to adsorb completely, the yield may be lowered; if it is lessthan 10 L·min⁻¹, it is disadvantageous for increasing the productivity.

[0061] After adsorption, the resin may be eluted by alcohol such asethanol or methanol, ethanol is preferred from the viewpoint of healthy.When using ethanol, the concentration of which is preferably 50-95%, andthe most preferred is 70% (may±about 5%) in consideration of thereutilization of ethanol.

[0062] While eluting, the ratio of the amount of alcohol to crude drugmay range from 2 to 10. If the ratio is less than 2, the elution isincomplete, if it is more than 10, the eluted components will notincrease further and is economically disadvantageous. In view ofmanufacturing, 2-5 folds is preferred.

[0063] After elution, the eluent is collected and concentrated afterremoving of ethanol for reutilize. Finally, brown or umber powder isobtained via spray-drying, lyophilizing or normal drying andpulverizing. The powder can be refined or directly filled into theproper preparations, such as capsules, pills, tablets, granules,solutions or injections.

[0064] Relative density of the residue is adjusted to 1.10-1.18 beforespray-drying, and is adjusted to 1.3-1.4 before vacuum drying. Afterdrying, the product may be pulverized to fine powder to facilitate thefurther processes. Spray-drying and vacuum drying are ordinarytechniques of drying and is not limited.

[0065] The Rhizoma Drynariae extract of present invention as describedabove may be prepared into any pharmaceutical preparations suitable forclinical uses, such as capsules, pills, tablets, granules, solutions orinjections upon ordinary preparation procedures. The dosage of each typeof preparation may vary depending on the composition of formulation,status of the patient and clinical conditions. General, in case when thecontent of total flavonoids in RDE is 50%, the oral administrationdosage may be 0.1-5.0 g/day in 1-4 portions per patient.

[0066] The Rhizoma Drynariae extract of present invention hasoutstanding activity of improving osteoporosis. The following Examplesare provided to illustrate the invention and do not limit it in any way.

EXAMPLES

[0067] The flavonoids in the RDE is qualitative characterized bypotassium borohydride (KBH₄) color-reaction. And the elution terminationis determined by thin layer chromatography (TLC). And ultraviolet (UV)spectrometry is applied to quantitative analysis of total flavonoids.High-performance liquid chromatography (HPLC) is applied toquantitatively analyze naringin.

[0068] Potassium borohydride color reaction: KBH₄ (about 5 mg) is addedinto a test tube containing about 5 ml of sample solution and shaken up,into which several drops of hydrochloric acid is added, the appearedcherry red or fuchsia shows the existence of dihydroflavonone(sensitivity 55 μg/ml). Otherwise it is negative if it doesn't appearcherry red or fuchsia.

[0069] TLC: Naringin standard in methanol to the concentration of 0.5mg/ml is used as control solution. Based on the TLC method in accordancewith Pharmacopoeia Sinica (1995) appendix VI B, each of 4 μl of controland test solutions are applied to one silica gel-G plate, after thateluted with the upper layer of the mixture of benzene-ethylacetate-formic acid-water (1:12:2.5:3). After elution, the plate istaken out and aired, sprayed by aluminum chloride (AlCl₃) solution andfinally determined under UV light at 365 nm. The test sample shows afluorescence blot same color as that of the control in the correspondingsite of the plate.

[0070] UV Spectrometry Quantitative Analysis of Total Flavonoids:

[0071] This analysis is based on the UV spectrophotometric method inPharmacopoeia Sinica (1995) appendix VA.

[0072] Preparation of control solution: 10 mg of Hesperidin standard,which is desiccated under 105° C. to constant weight, is preciselyweighed and dissolved in methanol (100 ml) in a volumetric flask (100ml). (Final concentration: 0.1 mg/ml)

[0073] Standard curve: Standard solutions of 0.50 ml, 1.00 ml, 2.00 ml,4.00 ml, and 5.00 ml are precisely pipetted and diluted with methanol to25 ml of volume respectively. Methanol is used as the blank control. UVdetection is performed in accordance with appendix VA), and absorbencyis determined at 284±1 nm. The calibration curve is plotted with theX-axis of concentration and the Y-axis of absorbency.

[0074] Sample solution preparation: testing sample (0.25 g), which isprecisely weighed, is dissolved in methanol (50 ml) in conical flask,ultrasonic extracted for 30 minutes, and transferred to a volumetricflask (100 ml) after cooling. And the conical flask is washed withmethanol the rinsing liquid is also transferred into the volumetricflask. And the solution is diluted by methanol to 100 ml, and thenfiltrated. The subsequent filtrate is collected, and a precise volume 1ml of the filtrate is applied to a chromatograph column (packed with 0.5g of polyamide as solid phase, which has passed 60 mesh of shieve). Thenthe column is eluted by methanol with a flow rate of about 0.4 ml/min.About 25 ml of eluent is collected and diluted by methanol to a volumeof 100 ml.

[0075] Measurement: According to the UV spectrophotometric method inPharmacopoeia Sinica (1995) appendix VA, the absorbency of the samplesolution is detected, and then the total flavonoids concentration isread from the standard curve.

[0076] Instruments and reagents: ultraviolet spectrometry, model UV-260(Shimazu Inc., Japan), hesperidin standard (from the National Institutefor the Control of Pharmaceutical and Biological Products)

[0077] HPLC Determination of Naringin

[0078] The test is based on the HPLC method of Pharmacopoeia Sinica(1995) appendix VI D.

[0079] Chromatographic conditions: C-18-alkylsilane-bonded-silica-gel isused as solid phase, a mixture of methanol-(36% acetic acid)-water(35:4:65) is used as mobile phase, detecting wavelength at 283 nm.Number of theoretical plates is calculated from the peak of naringin,and should be no less than 3000.

[0080] Preparation of control solution: Naringin standard, which isdesiccated under 105° C. to constant weight, is precisely weighed anddissolved in methanol and diluted with methanol to the finalconcentration 50 μg/ml.

[0081] Preparation of sample solution: the product (ca. 0.2 g) isprecisely weighted and put into 150 mL conical flask, 60 mL of methanolis added therein. After 30 mins of ultrasonic extraction, the solutionis taken out and cooled to room temperature. Afterwards the samplesolution is transferred into a 100 mL volume volumetric bottle. Theconical flask is washed with methanol and methanol is combined into thebottle. The obtained solution is diluted to 100 ml by methanol, shakedto uniform and filtrated. The subsequent filtrate is collected, 2 ml ofthe filtrate is precisely pipetted into a 25 ml volumic bottle anddiluted with methanol to the volume to obtain the sample solution.

[0082] Measurement: Control solution (10 μl) and sample solution (10 μl)is respectively injected into the HPLC system for analysis.

[0083] Instruments and reagents: HPLC: LC-6A (Shimazu Inc., Japan); UVdetector: SPD-6AV; data processor (C-R4A), sensibility of data recorder0.16AUFS; Chromatograph Column: Shimpack C₁₈ column (CLC-ODS 150×6.0 mmi.d., 5 μm); Protecting Column: YMG C₁₈ column (10×4.6 mmi.d., 10 μm);Supercentrifuge: TGL-16G (Shanghai Medical Analytical InstrumentsFactory); Ultrasonic cleaner: SB 3200 (Shanghai Bi'nengxin UltrasonicCompany, Ltd)

[0084] Standards of naringin are obtained from National Institute forthe Control of Pharmaceutical and Biological Products. Under the abovespectrum conditions, it is calculated that naringin contained in thepresent standards is more than 98% by normalized method (see FIG. 1).Under the present experimental conditions, the peak of naringin isappeared at the retention time of about 15.26 min.

[0085] All the reagents and solvents used in the examples areanalytically pure grade. Water is double-distilled.

Example 1

[0086] 100 grams of Rhizoma Drynariae was pulverized and decocted in1500 ml of water for 1 hour, the extract liquid was removed andadditional 1000 ml of water was added and detocted again. The twodetoction liquids were combined and filtrated. The filtrate was appliedto a pretreated column (φ 30 mm) packed with 120 g of WLD resin at aflow rate of 4 ml/min, and the terminated point of effluent collectionwas determined by KBH₄ color reaction, that is, the sudden appearance ofdihydroflavonone showed the saturation of adsorption in theeffluent-resin. After adsorption, the column was washed with 500 ml ofwater and washing liquid was not-recycled. Elution was conducted with400 ml of 70% ethanol. The starting and end point of elution wasdetermined by KBH₄ color test, eluent was collected and ethanol wasrecycled. Residual was evaporated on a water bath to give thick paste,which was then dried in vacuo to afford 1.88 g of extract. Thus obtainedRDE had a total flavonoids content of 55.08% detected by UVspectrometry, and a naringin content of 36.6% (i.e. 66.4% of the totalflavonoids) determined by HPLC.

[0087]FIG. 2 shows the UV spectrum of the solutions of RDE, naringin,and blank control (X-axis: wave length; Y-axis: absorbency; 1: naringinstandard; 2: present extract; 3: blank). From the UV spectrum, greatdifferentiation can be observed between RDE and naringin. RDE has anabsorption peak at around 200 nm, whereas naringin has only a shoulderpeak at that wavelength. And there is also difference in the maximumabsorption wavelength.

[0088] The high-performance liquid chromatogram of the extract is shownin FIG. 3, in which there is at least one another unknown compoundco-existing in the extract. (X-axis: Retention time; Y-axis: Integralintensity).

Example 2

[0089] In the same manner as that of Example 1 except that 70% ofethanol was used for reflux and elution solvent. A 1.69 g of extract wasobtained, in which the total flavonoids content was 53.12%, and naringinis 32.5% determined by HPLC (61.2% of the total flavonoids).

[0090] The comparison of TLC for several extracting solutions extractedby different processes are shown in FIG. 4, in which from left to rightare chromatograms of extractant and extract of example 1, extractant andextract of present example, and naringin. From the results can we seethat the extracts are substantially same no wonder extracted by water oralcohol.

Example 3

[0091] In the same manner as that of Example 1 except that AB-8 resinwas used instead of WLD resin. A 0.95 g of extract was obtained, withthe total flavonoids content of 61.80%. FIG. 5 shows the HPLC of theextract of Example 3.

Example 4

[0092] In the same manner as that of Example 1 except that D₁₀₁ resinwas used instead of WLD resin. A 1.07 g of extract was obtained, withthe total flavonoids content of 57.35%. FIG. 6 shows the HPLC of theextract of in Example 4.

[0093] From FIGS. 3, 4 and 5 can we see that although different types ofresin was used as the adsorbent, the composition and content ratio offlavonoids in the extracts are generally the same.

Example 5

[0094] In the same manner as that of Example 1 except that 10 g ofRhizoma Drynariae power was used each time, and 5 g, 10 g, 20 g and 25 gof resins were used respectively. 0.16 g, 0.20 g, 0.27 g and 0.27 g ofthe extract were obtained accordingly, wherein the total flavonoidscontents are 53.88%, 52.29%, 42.65% and 41.88%, respectively. The yieldof total flavonoids were 86.2 mg, 104.6 mg, 115.2 mg and 113.1 mg,respectively, and the total flavonoid ratio are 0.86%, 1.05%, 1.15% and1.13%, respectively. The above data shows that the total flavonoidsextracted from the same amount of crude drug is almost not increasingwhen the weight ratio of crude drug to resin is more than 1:2, whichmeans that the adsorption is complete when the resin is 2× the amount ofthe crude drug. When weight ratio of crude drug to resin is 1:1, thetotal flavonoids in adsorbate is relatively higher: the extract has anis amount of 90.8% of the full adsorption, with the total flavonoidscontent of 52.29%. Therefore, a ratio at about 1:1 of crude drug: resinis preferred.

Example 6

[0095] A 40 g of pulverized Rhizoma Drynariae was decocted with 1000 mlof water for 1 hour (slow fire), and the decoctate was filtrated anddivided into four portions averagely. Four glass columns (φ 20 mm) werepacked with pretreated WLD macropore adsorption resin (10 g). Everyportion of decoctates was applied to one column respectively atdifferent flow rates of 2 ml/min, 4 ml/min, 8 ml/min and 16 ml/min, andKBH₄ color reaction was applied to test whether there was flavonoidglycoside in the effluent. Results are shown in table 1. TABLE 1 Flowrate (ml/min) Effects Flavonoid in effluent 2 Completely adsorbed − 4Completely adsorbed − 8 Incompletely adsorbed + 16 Incompletely adsorbed+

[0096] It can be concluded that the control of flow rate ensurescomplete adsorption.

Example 7

[0097] A 300 g of raw pulverized Rhizoma Drynariae was treated in thesame manera as Example 1 to obtain the filtrate. And the filtrate wasdivided to three portions averagely, each was applied to the column (φ40 mm, packed with 100 g of WLD resin), then eluted with ethanol ofdifferent concentrations. The eluent was concentrated and dried, thetotal flavonoids contents were measured via UV spectrometry. The resultsare shown in table 2. TABLE 2 Ethanol concentration (%) (Group) 0% 50%(1) 70% (2) 95% (3) Total flavonoids 0 1.668 1.888 1.587 (g) 0 1.6721.843 1.531 0 1.679 1.871 1.574 {overscore (x)} ± SD 0 1.673 ± 0.0061.876 ± 0.023 1.564 ± 0.024

[0098] It can be concluded that the concentration of ethanol affects theeffect of elution, in which the concentration of about 70% is preferred.

Example 8

[0099] Raw powder of Rhizoma Drynariae (500 g) was treated as the samemanner that of Example 1 to obtain the filtrate, which then is appliedto a column (weight ratio of crude drug to resin is 1:1). Afteradsorption, the resin was eluted with 70% ethanol in the volume of 1×,2×, 3×, 5×, 7× and 10× amount of crude drug (v/w), the eluents werecollected separately, from which the ethanol was recycled. Then thetotal flavonoids was measured separately. The results show that theelution is complete when elution by 10 folds of ethanol, which is shownin table 3. TABLE 3 70% ethanol 1x 2x 3x 5x 7x 10x Sorbent (%) 0.47 8.761.99 0.47 0.44 0.22 Flavonoids 0.21 53.11 64.26 51.42 24.87 6.72 (%)Flavonoids 0.987 4652.436 1278.774 241.674 109.428 14.784 (mg) % oftotal 0.016 73.871 20.304 3.837 1.737 0.235 flavonoids

[0100] Results in table 3 show that the total flavonoids can be elutedcompletely, when eluted by enough quantity of ethanol (10 folds). Whenthe quantity of ethanol is 5 folds of RD, from which the totalflavonoids is amounted to 98% of the total flavonoids.

Preparation Example 1, Capsule

[0101] To 180 g powder of RDE obtained in the same manner that ofExample 1, 70 g of starch was added. The two components were mixed welland then filled into 1000 capsules. Each capsule contains 180 mg ofeffective component.

Preparation Example 2, Tablet

[0102] To 180 g powder of RDE obtained in the same manner that ofExample, 70 g of starch was added. Then the mixture was kneaded with asmall amount of aqueous CMC-Na and then pelletized, dried according tothe ordinary methods, into which a small amount of magnesium stearatewas added and mixed well to produce 1000 tablets. Each tablet contains180 mg of effective component.

Preparation Example 3, Injectable Formulation

[0103] To 180 g powder of RDE obtained in the same manner that ofExample, 800 ml of injectable distilled water was added. The resultantmixture was heated to solve, then filtrated by microporous filtermembranes, stored for 24 hours at 4° C.-8° C., then filtrated again,adjusted pH to 6.5 and isotonic with medicinal grade aqueous HCl (12N)and NaOH (12N), finally injectable distilled water was added upto 1000ml, sterilized by heating, and filtrated, then divide into ampoules (5ml per ampoule). Each ampoule contains 180 mg of effective component.

Test Example 1. The Clinical Test of RDE of Present Invention on PrimaryOsteoporosis.

[0104] The drugs with the following dosages and contents are used unlessadditional explanations.

[0105] Extracts in this present invention: capsules of preparationexample 1, each capsule contains 0.18 g of effective component. Positivecontrol medicine: α-D₃, (synthesized by TEVA PHARMACEUTICALS, Israel,prepared by Sino-US Kunming BARKER NORTON PHARMACEUTICAL CO., LTD), eachcapsule contains 0.5 μg of effective component)

[0106] Diagnostic criteria: Comprehensive Diagnostic Grades for PrimaryOsteoporosis, according to the diagnostic criteria established bySpecialized Committee of Osteoporosis of Chinese Association ofGeriatrics. (cf. Gu-zhi-shu-song-zheng [M], p169-171, LIU Zhonghou eds.Chemical Industrial Press, Guangzhou, 1992)

[0107] According to the diagnostic criteria mentioned above, 70 cases ofosteoporosis are randomly divided into treatment group with RDE andcontrol group with α-D₃. RDE is administrated 180 mg, per os, tid, for 6months-2 period of treatment. And α-D₃ is administrated 0.5 μg, per os,bid, for 6 months too.

[0108] Bone density of lumbar (L₂-L₄) and top-femur (collum femoris,trigonal area of femur, trochanteric of femur) is checked respectivelyby Double-Energy X-ray Bone Density Meter (LUNAR DPX-L DEXA bonedensitometer,US). The unit is g/cm² . And an Ultrasonic BoneDensitometer (model 2000-SYS-220, METRA Co., Israel) is employed tocheck the bone density of mid-portion of tibia.

[0109] The comparison of bone density pre- and after-treatment in twogroups:

[0110] 1. The results of bone density of lumber (L₂-L₄) pre- andafter-treatment are shown in table 4: TABLE 4 Bone density of lumber(L₂-L₄) before- and after- treatment in two groups (x ± s, g/cm²)Before- after- 3 months after- 6 months Group treatment treatmenttreatment Treatment group (17) 0.973 ± 0.124 1.038 ± 0.234 1.101 ± 0.303Control group (17) 0.958 ± 0.180 0.954 ± 0.179 0.958 ± 0.145

[0111] The bone density increased after 3 or 6 months treatment fortreatment group, but it is of no significant difference (P>0.05). Thereis no improvement in control group. Also there is no significantdifference between the two groups.

[0112] 2. The results of bone density of collum femoris pre- andafter-treatment are shown in table 5: TABLE 5 Bone density of collumfemoris before- and after- treatment in two Groups (x ± s, g/cm²) after-3 months after- 6 months Group Before-treatment treatment treatmentTreatment 0.736 ± 0.079  0.750 ± 0.079**  0.828 ± 0.124** group Control0.748 ± 0.095 0.744 ± 0.100 0.744 ± 0.093 group

[0113] There is significant increase in bone density of collum femoriscompared between after- and before-treatment within treatment group(P<0.05, P<0.01). But it is of no significant difference within controlgroup.

[0114] 3. The results of bone density of trigonal area of femur pre- andafter-treatment are shown in table 6: TABLE 6 Bone density of trigonalarea of femur before- and after- treatment in two Groups (x ± s, g/cm²)after- 3 months After- 6 months Group Before-treatment treatmenttreatment Treatment 0.585 ± 0.104  0.600 ± 0.100*   0.661 ± 0.159**^(#)group Control 0.582 ± 0.125 0.591 ± 0.124 0.581 ± 0.134 group

[0115] There is significant increase in bone density of trigonal area offemur compared between after- and before-treatment within treatmentgroup (P<0.05, P<0.01). The bone density of trigonal area of femurincreased after 3 months of treatment with no significant difference(P>0.05). And also there is difference between treatment and controlgroups after 6 months treatment (P<0.05).

[0116] 4. The results of bone density of trochanteric of femur pre- andafter-treatment are shown in table 7: TABLE 7 Bone density oftrochanteric of femur pre- and after- treatment in two Groups (x ± s,g/cm²) after- 3 months after- 6 months Group Before-treatment treatmenttreatment Treatment 0.642 ± 0.120 0.665 ± 0.125  0.752 ± 0.175** groupControl 0.606 ± 0.109 0.616 ± 0.099 0.616 ± 0.094 group

[0117] There is significant difference between before- andafter-6-months-treatment in treatment group (P<0.01). And also there issignificant difference between treatment and control groups after 6months treatment (P<0.01).

[0118] 5. The results of ultrasonic bone density of pre- andafter-treatment in two groups are shown in table 8: TABLE 8 ultrasonicbone density in two groups (X ± s, g/cm²) Before- after 3 months after 6months Item Group n treatment treatment treatment Young Treatment 16−1.875 ± 0.703    −1.643 ± 0.766**  −1.288 ± 0.857** adult Control 15−1.993 ± 0.872   −1.953 ± 1.181   −0.593 ± 1.209   Age Treatment 16−1.400 ± 0.700    −1.200 ± 0.701**#  −0.956 ± 0.682**# matched Control15 −1.433 ± 0.788   −1.386 ± 0.908   −0.987 ± 1.068   Speed Treatment 163769.42 ± 77.61   3793.00 ± 81.81**#  3836.33 ± 99.03**# sound control15 3770.51 ± 89.45  3761.77 ± 129.66* 3804.48 ± 138.89 

[0119] The difference values between bone density (ultrasonic) oftreatment group and those of young adults and age matched increasedsignificantly (P<0.01). And the speed sound increase significantlyself-compared within treatment group (P<0.01) and within control group(P<0.05).

Test Example 2, Experimental Study of RDE on Osteoporosis Models in Rats

[0120] Observe the effect of RDE in this invention on bothTretinoin-induced and Ovariectomized (OVX) osteoporosis model in rats.

[0121] [Material and Methods]

[0122] 1. Drugs

[0123] RDE was produced according to Example 1 (Per gram extract isequivalent to 66.67 g crude drugs). RDE was dissolved in distilled waterto a concentration of 0.47 mg/ml before administration. Andadministration was conducted via gastric tube. Tretinoin is manufacturedby Chongqing Huabang Pharmaceuticals, Ltd. and was suspended to theconcentration of 1.5% with distilled water. Control drug α-D₃ wasmanufactured by TEVA PHARMACEUTICALS, Israel, which is solved indistilled water to the concentration of 0.03 μg/ml before using andadministrated orally via gastric tube.

[0124] 2. Animals

[0125] Tretinoin-induced osteoporosis rats: sixty female W istar rats,16-week-old, mean weight 234.85±15.32 g. Ovariectomized rats: sixtyfemale Wistar rats, 12-week-old, mean weight 183.46±12.45 g. All theanimals are obtained from the Experimental Animal Center of ChineseMilitary Medical Academy.

[0126] 3. Reagents and Instruments

[0127] Bone Biomechanics Rheometer was manufactured by Stevens Company,Great Britain (model QTS-25). X ray Bone-density-meter was manufacturedby Norland Company, USA (model XR-26). The others are all obtained frommarket.

[0128] 4. Dose Selections and Groups:

[0129] (1) Tretinoin-induced group: Sixty rats are randomly allocated tosix groups (10 rats per group). Blank group: control animals receivedPSS/d (physiological saline solution) alone. Model group: Tretinoin isgiven orally 70 mg/kg/d; α-D₃ group: both tretinoin and α-D₃ are givenorally 70 mg/kg/d and 0.002 μg/kg/d respectively; RDE low-dose group:both tretinoin and RDE are given orally 70 mg/kg/d and 54 mg/kg/drespectively; RDE medium-dose group: tretinoin 70 mg/kg/d and RDE 108mg/kg/d; RDE high-dose group: tretinoin 70 mg/kg/d and RDE 216 mg/kg/d.

[0130] (2) Ovariectomized (OVX) rats group: Sixty rats are randomlyallocated to six groups (10 rats per group). Blank group is the normalrats. All the other groups are removed amphi-ovaries. Model groupreceived PSS only; α-D₃ group: α-D₃ is administrated orally 0.002μg/kg/d; And RDE is administrated orally in the dose of 54, 108, 216mg/kg/day to OVX rats respectively, corresponding to RDE groups oflow-dose, medium-dose and high-dose.

[0131] 6. Methods:

[0132] The dose administered to each animal is calculated from theindividual body weight recorded at the end of each week.

[0133] Tretinoin-induced osteoporosis groups: Blank group received PSSduring the course of experiment. The other groups commenced treatment onthe same day, designated study Day 1, and once-daily tretinoin orallygiven for 14 days, after which they stopped administrating tretinoin.Both blank and model groups are given PSS in Day 15, and α-D₃ group isgiven α-D₃, RDE groups are given the corresponding dose of RDE orallyfor 14 days. The final dose is given in the Day 28 before sacrificed.Investigation of Bone Density and Bone-biomechanics are performed in theDay 29.

[0134] OVX rats groups: Both blank and model groups are given the samevolume of distilled water orally, All the other groups commencedtreatment according to the way mentioned above for 12 weeks after whichanimals are sacrificed and investigation of Bone Density andBone-biomechanics are performed.

[0135] 7. Measurements

[0136] 7.1. Bone density measurements are performed on No.2-4 lumbarspines and right thighbone of each animal.

[0137] 7.2. Bone biomechanics measurements: three point bending tests ofleft thighbone of each animal are performed; load-deformation curve isrecorded to measure the biomechanics index. Experimental parameters:span 20 mm, loading velocity 10 mm/min. Bone structural mechanicsparameters: breaking load, elastic load, maximum radius, elastic radius,maximum energy absorption; bone material mechanics parameters: firstlycalculated the Femoral Section Inertial Moment, then computed themaximum strain, elastic strain, maximum stress, elastic stress andelastic modulus according to the formulae.

[0138] [Results]

[0139] 1. Bone Density of Rats in Each Group (Table 9 and 10) TABLE 9Bone density of Tretinoin-induced osteoporosis in rats Bone density(g/cm²) Group n thighbone Lumber spine Blank 10 0.146 ± 0.008 0.147 ±0.005 Model 10 0.124 ± 0.006 0.129 ± 0.00  α-D₃ 10 0.137 ± 0.004 0.135 ±0.005 RDE low-dose 10 0.140 ± 0.005 0.135 ± 0.005 RDE medium-dose 100.140 ± 0.007 0.149 ± 0.007 RDE large-dose 10 0.152 ± 0.006 0.149 ±0.011

[0140] TABLE 10 Bone density of Ovariectomized (OVX) rats group Bonedensity (g/cm²) Group n thighbone Lumber spine Blank 10 0.14 ± 0.0080.13 ± 0.009 Model 10 0.11 ± 0.007 0.11 ± 0.035 α-D₃ 10 0.13 ± 0.0090.13 ± 0.009 RDE low-dose 10 0.12 ± 0.005 0.12 ± 0.005 RDE medium-dose10 0.12 ± 0.005 0.12 ± 0.010 RDE large-dose 10 0.13 ± 0.007 0.13 ± 0.004

[0141] From Table 9 and 10 it is concluded that femoral bone density ofRDE groups and α-D₃ group increased obviously compared with model group.And there is significant differences in elevated degree of femoral bonedensity compared between RDE groups and α-D₃ group.

[0142] 2. Bone Biomechanics Measurements of Rats in Each Group

[0143] 2.1 The influence on bone structural mechanics indicators, seetable 11 and Table 12. TABLE 11 Bone structural mechanics indicators ofRetinoic acid-induced osteoporosis in rats Maximum load Maximum strainMaximum energy observation Elastic strain Group n (N) (mm) (N-mm)Elastic load (N) (mm) Blank 10 146.60 ± 30.80 1.54 ± 0.36 787.95 ±130.57 159.10 ± 88.80 1.65 ± 0.20 Model 10  66.10 ± 11.00 0.64 ± 0.10405.88 ± 160.06  53.60 ± 19.80 0.44 ± 0.18 α-D₃ 10 103.20 ± 3.40  0.98 ±0.14 635.73 ± 114.46 100.40 ± 7.00  1.01 ± 0.14 RDE low-dose 10 123.60 ±6.09  1.04 ± 0.13 564.38 ± 138.89 105.70 ± 4.30  1.06 ± 0.09 RDEmedium-dose 10 135.30 ± 12.00 1.33 ± 0.22 655.13 ± 131.46 110.00 ± 12.601.56 ± 0.19 RDE large-dose 10 145.80 ± 11.00 1.65 ± 0.25 769.62 ± 128.43120.40 ± 8.70  1.75 ± 0.16

[0144] TABLE 12 Bone structural mechanics indicators of Ovariectomized(OVX) rats Maximum strain Elastic strain Maximum energy Group N Maximumload (N) (mm) Elastic load (N) (N) observation (N-mm²) Blank 10 118.3 ±12.40 1.11 ± 0.05 96.2 ± 6.00 0.93 ± 0.05 575.09 ± 138.50 Model 10  76.8± 25.00 0.68 ± 0.01 62.5 ± 2.50 0.48 ± 0.07 425.25 ± 86.10  α-D₃ 10102.1 ± 2.90  0.96 ± 0.10 88.6 ± 12.7 0.84 ± 0.06 520.48 ± 108.44 RDElow-dose 10 97.0 ± 6.50 0.71 ± 0.10 72.1 ± 7.80 0.67 ± 0.09 498.55 ±162.80 RDE medium-dose 10 110.4 ± 10.20 0.92 ± 0.06 91.6 ± 6.70 0.85 ±0.06 543.65 ± 100.78 RDE large-dose 10 134.3 ± 9.50  1.10 ± 0.11 99.4 ±4.60 0.93 ± 0.05 532.37 ± 110.83

[0145] From above table 11 and 12, the results showed that all the bonestructural mechanics indicators in RDE groups and α-D₃ group increasedsignificantly compared with model group. Also there is significantdifferences in the elevated degree of all the bone structural mechanicsindicators compared between RDE groups and α-D₃ group. And we can seesome dose-effect relationship in RDE groups.

[0146] 2.2 The effect on bone material mechanics indicators, see table13 and 14. TABLE 13 Bone material mechanics indicators ofTretinoin-induced osteoporosis in rats Maximum stress Elastic stressRigidity coefficient Group n (N/mm²) (N/mm²) Maximum strain Elasticstrain (N/mm²) Blank 10 309.3 ± 102.8 233.2 ± 48.5  0.04 ± 0.009 0.04 ±0.013 41301 ± 1359 Model 10 122.24 ± 54.8  70.9 ± 20.5 0.02 ± 0.004 0.01± 0.004 14436 ± 2648 α-D₃ 10 167.2 ± 44.5  177.6 ± 51.3  0.03 ± 0.0090.03 ± 0.01  30561 ± 9194 RDE low-dose 10 101.8 ± 3.1  79.9 ± 21.1 0.04± 0.004 0.02 ± 0.005 14958 ± 2347 RDE medium- 10 236.1 ± 59.2  217.5 ±79.2  0.03 ± 0.004 0.03 ± 0.011 24919 ± 2616 dose RDE large-dose 10307.9 ± 7.9  333.9 ± 27.1  0.04 ± 0.001 0.04 ± 0.007 36893 ± 3704

[0147] TABLE 14 Bone material mechanics indicators of Ovariectomized(OVX) rats Maximum stress Elastic stress Rigidity Maximum Elastic Areaof bone Group N (N/mm²) (N/mm²) coefficient strain Elastic strain mouldcortes Blank 10 254.2 ± 32.50 197.5 ± 22.40 44383 ± 3022 0.04 ± 0.0030.04 ± 0.003 9516 ± 267 6.23 ± 0.52 Model 10 123.4 ± 43.50 99.7 ± 8.60 34620 ± 11208 0.03 ± 0.002 0.01 ± 0.002 4742 ± 748 3.53 ± 0.56 α-D₃ 10221.5 ± 46.50 147.2 ± 11.00 27508 ± 8073 0.03 ± 0.006 0.03 ± 0.010  7992± 1132 4.13 ± 0.99 RDE 10 138.3 ± 41.80 151.1 ± 12.50 33114 ± 4718 0.04± 0.004 0.03 ± 0.006 7233 ± 872 4.02 ± 0.07 low-dose RDE medium- 10140.0 ± 40.60 137.7 ± 10.50 32729 ± 3307 0.05 ± 0.002 0.04 ± 0.002 7546± 613 4.68 ± 0.44 dose RDE 10 249.9 ± 18.70 208.7 ± 17.80 44019 ± 19990.05 ± 0.003  0.04 ± 0.0.04 8768 ± 543 6.15 ± 0.37 large-dose

[0148] From table 13 and 14, the results showed that RDE elevated thebone maximum stress, elastic stress, maximum strain, elastic strain andrigidity factor of osteoporosis model rats. And there is significantdifference in those indicators compared between RDE group and α-D₃group.

[0149] From the results mentioned above, we can see that RDE elevate thebone density, enhance bearing capacity and anti-impact capacity toimprove the bone structural mechanics properties, and also has activityto improve geometric configuration and inner material properties.

Industrial Practicability

[0150] RDE in the present invention has the activity to elevate the bonedensity of osteoporosis patient, to suppress the bone absorptiondistinctly, and of anti bone-loss. Thereafter the object of treatingosteoporosis can be achieved.

What is claimed is:
 1. An extract from Rhizoma Drynariae, wherein thecontent of total flavonoids, based on the weight of the extract, is 30percent or more, and the content of naringin thereof, based on theweight of total flavonoids, is 30 percent or more, and less than 100percent.
 2. The extract according to claim 1, wherein the content oftotal flavonoids is 50 percent or more.
 3. The extract according toclaim 2, wherein the content of naringin is 90 percent or less.
 4. Apharmaceutical composition comprises the extract of claim
 1. 5. Thecomposition according to claim 4, which is used for the treatment ofOsteoporosis.
 6. Use of the extract according to claim 1 in thepreparation of medicaments for the treatment of osteoporosis.
 7. Use ofthe extract according to claim 1 for the treatment of osteoporosis.
 8. Apreparation process of the extract according to claim 1, comprising: 1)extraction Rhizoma Drynariae with water, alcohol or a mixture thereof;2) adsorption of the extractant with resin; and 3) elution of the resinwhich has absorbed the extractant with water, alcohol or a mixturethereof.
 9. The preparation process according to claim 8, wherein theextraction is conducted with water.
 10. The preparation processaccording to claim 8, wherein the extraction is conducted withalcohol/water mixture.
 11. The preparation process according to claim 8or 10, wherein the content of alcohol in the alcohol/water mixture is40-90%(wt %).
 12. The preparation process according to claim 8, 10 or11, wherein the alcohol is selected from methanol and ethanol.
 13. Thepreparation process according to claim 12, wherein the alcohol isethanol.
 14. The preparation process according to claim 13, wherein theresin is macropore adsorption resin.
 15. The preparation processaccording to claim 8 or 14, wherein said resin is washed by water afteradsorption of the extratant, before the elution with alcohol oralcohol/water mixture.
 16. The preparation process according to claim15, wherein the extraction is conducted with alcohol/water mixture. 17.The preparation process according to claim 15 or 16, is wherein thecontent of alcohol is 40-90% (w/w) of the alcohol/water mixture.
 18. Thepreparation process according to claim 15, 16 or 17, wherein the alcoholis selected from methanol and ethanol.
 19. The preparation processaccording to claim 18, wherein the alcohol is ethanol.